We have expanded our biophysical/biochemical studies of modifications in P. falciparum-infected erythrocytes to include (1) Zeta potential measurements, (2) detergent-resistant membrane studies, and (3) lipid analyses of HbA and HbC erythrocytes. Zeta potential is a measure of cell-surface electrochemical potential and preliminary data indicate that it changes during the course of a malaria infection. In a continuation of our band 3 clustering study demonstrating that both uninfected and infected CC erythrocytes showed increased band 3 clustering over AA erythrocytes which may responsible for enhanced CC protection against severe malaria, we found a clear difference exists in zeta potential: CC erythrocytes have a zeta potential of ~ -13 mV whereas AA erythrocytes have a zeta potential of ~ -15 mV. This difference may be consequence of (1) serum protein binding to the erythrocyte surface, (2) protein clustering which may be partially induced by hemichrome aggregation, and/or (3) lipid modification due to hemoglobin degradation-induced oxidation. [unreadable] To better understand these data we are comparing membrane rafts, or detergent-resistant membrane structural differences between AA and CC erythrocytes. We found that the distribution pattern of flotillin-1, one of the major raft makers, shifted to denser fractions in a sucrose gradient. Most of flotillin-1 appeared in raft fractions. In contrast, band 3, which is a non-raft marker, showed a mixed distribution: some of the band 3 appeared in raft fractions, but the majority of band 3 appeared in non-raft fractions. We found the same pattern for CD 47, which has a negative control effect on erythrocyte recycling by macrophages. The distribution patterns in CC erythrocytes for these two proteins also appeared to shift towards denser fractions. The major erythrocyte structural protein spectrin did not appear in raft fractions in either AA or CC erythrocytes. These data showed that the CC erythrocyte membrane is, in general, denser and the protein actually involved in erythrocyte-recycling signals exists in a different state which may be directly related to the higher protection effect against severe malaria of CC erythrocytes. Lipid analysis of CC cells is on-going using a variety of techniques such as TLC and HPLC. [unreadable] Following the transfer of Dr. Ostera to our laboratory, we began studies of the synthesis and utilization of nitrogen-containing compounds by malaria parasites. The study is multifaceted and includes the characterization of the biosynthetic pathway(s) used by P. falciparum to produce nitric oxide as well as the biological impact of nitric oxide produced by the parasite in the pathophysiology of cerebral malaria. [unreadable] Expansion of applied AFM studies show that cross-linking of the erythrocyte membrane inhibits invasion but does not inhibit knob formation in an erythrocyte infected prior to treatment.